Analysis of nucleic acids requires, according to various modalities of recognition, preliminary steps of preparation of a specimen of biological material, amplification of the nucleic material contained therein, and hybridization of individual target or reference strands, corresponding to the sequences sought.
At the end of the preparatory steps, the specimen is examined to check whether amplification has regularly occurred.
According to a methodology referred to as “real-time PCR”, the DNA is amplified through appropriately selected thermal cycles, and the evolution of the amplification reactions is detected and monitored by fluorescence throughout the process.
The amplification reactions are conducted in such a way that the strands, which are contained in a recognition chamber provided in a support, include fluorescent molecules or fluorophores. PCR analysers designed to be used for optical reading of the specimens are described in U.S. Patent Application Publications 2012/0170608 and 2013/0004954.
There is a need to provide chips having recognition chambers that have a hydrophilic base portion and a hydrophobic lateral-edge portion. This need is achieved, according to the state of the art, by providing on a chip with a hydrophilic surface a containment structure of hydrophobic material, which defines a plurality of chambers.
In the absence of a hydrophobic confinement, the solution contained in the chambers that includes the biological specimen and the reagents, may assume an excessively peripheral distribution, both at the expense of conditions of reaction, the uniformity of which may be jeopardized to the extent of markedly slowing down or even preventing the reaction altogether, and at the expense of the external detection of the signal.
Consider the case where arranged on a chip, which is provided with a surface of silicon oxide and possibly further treated to improve the hydrophilic nature thereof, is a polycarbonate structure that borders on the reaction chambers. Let moreover each of these chambers be provided with heaters and temperature sensors, or other types of actuators and sensors. Let the system further include external systems for detection of signals that are indicative of the evolution of the reaction itself. It is clear that, in such a configuration, these systems are most effective at the central area of the chambers.
For instance, the heaters will be arranged centrally and will control the temperature with the maximum accuracy in the central area of the drop of fluid in which the reaction occurs, whereas an approximately radial gradient of temperature is to be expected.
In the case where the radial area is affected by a concentration of a considerable percentage of the fluid on account of the insufficient hydrophobicity of the polycarbonate, this may represent a serious obstacle to the proper conduct of the reaction, above all in the case of biological reactions that are very sensitive to the temperature, such as DNA amplification by PCR. Furthermore, in the same example, the sensors for detection of the fluorescence signal will be focused on the central area, where in the case of insufficient volume of fluid the quantitative signal detected might be weakened, causing errors in the estimation of the amount of DNA obtained by PCR.
For instance, it is known to use reactors for generation of a plasma in order to render the polycarbonate surface hydrophobic.
For this purpose, it is, however, expedient to mask the bottom of the reaction chambers in such a way as to preserve the hydrophilic characteristics thereof, or in any case not render them hydrophobic. For this purpose, it is common to use metals, such as aluminium, nickel or chrome, or compounds such as aluminium nitride (AlN) or aluminium oxide (Al2O3), or again silicon carbonate (SiC) or tantalum pentoxide (Ta2O5) as the mask. Reference may be made, for example, to Sami Franssila, “Introduction to microfabrication”, John Wiley & Sons, Second Edition, p. 138.
The mask is obtained by depositing a metal layer within the reaction chambers for covering the bottom of the chambers themselves by successive steps of deposition and etching, and then proceeding with plasma treatment for rendering the lateral surface of the chambers, which have not been masked, hydrophobic. Finally, the metal mask layer is then removed.
It is evident that the prior art poses serious problems of compatibility and process. In particular, the formation of a metal mask is undesirable because it complicates and lengthens the process steps, and may be a source of contamination for possible electronic devices integrated in the chip itself.